Surface Sialic Acid Detection of Small Extracellular Vesicles at the Single-Particle Level by Nano-Flow Cytometry

Glycans, particularly sialic acids (SAs), play crucial roles in diverse biological processes. Despite their significance, analyzing specific glycans, such as sialic acids, on individual small extracellular vesicles (sEVs) has remained challenging due to the limited glycan capacity and substantial heterogeneity of sEVs. To tackle this issue, we introduce a chemical modification method of surface SAs on sEVs named PALEV-nFCM, which involves periodate oxidation and aniline-catalyzed oxime ligation (PAL), in conjunction with single-particle analysis using a laboratory-built nano-flow cytometer (nFCM). The specificity of the PALEV labeling method was validated using SA-decorated liposomes, enzymatic removal of terminal SA residues, lectin preblocking, and cellular treatment with an endogenous sialyltransferase inhibitor. Comprehensive mapping of SA distributions was conducted for sEVs derived from different sources, including conditioned cell culture medium (CCCM) of various cell lines, human saliva, and human red blood cells (RBCs). Notably, treatment with the calcium ionophore substantially increases the population of SA-positive RBC sEVs and enhances the SA content on individual RBC sEVs as well. nFCM provides a sensitive and versatile platform for mapping SAs of individual sEVs, which could significantly contribute to resolving the heterogeneity of sEVs and advancing the understanding of their glycosignature.